臺灣博碩士論文加值系統

本研究利用石墨烯量子點（GQDs）與人類免疫缺陷病毒修飾辣根過氧化物酶的二級抗體（HRP-HIV-Ab2）生物綴合(bioconjugate)後製成新型電化學免疫感測器，用於檢測人類免疫缺陷病毒p24（HIV-p24）。具有活性結合位點(active binding sites)的GQDs提供了有效的生物界面來控制HRP-HIV-Ab2的負載量，因此顯著增強了此電化學感測器的敏感性。本研究採用三明治免疫分析方法(sandwich-type)將人類缺陷免疫病毒抗體與抗原固定於電極上，透過人類免疫缺陷病毒之二級抗體上的辣根過氧化物酶與溶液中的對苯二酚(hydroquinone)與過氧化氫(hydrogen peroxide)反應，達到顯著的信號放大效果。此製備的電化學免疫感測器顯示出優異的偵測極限（1.2 pg mL-1），線性範圍（0.00975 - 5 ng mL-1）和良好的選擇性，證明了HIV感染臨床診斷的有希望的潛在應用。
關鍵字：電化學免疫感測器；石墨烯量子點；人類免疫缺陷病毒 p24

A new electrochemical immunosensor based on the bioconjugation of graphene-quantum dots (GQDs) and horseradish peroxidase-labeled secondary antibody (HRP-HIV-Ab2) was designed for detecting human immunodeficiency virus p24 (HIV-p24). GQDs with active binding sites provided an efficient biointerface to control the loading amount of HRP-HIV-Ab2. Thus, the sensitivity of the electrochemical immunosensors was remarkably enhanced by employing the HRP-HIV-Ab2 linked to functionalized GQDs bioconjugates (HRP-HIV-Ab2-GQDs). With a sandwich-type immunoassay format, the HRP-HIV-Ab2-GQDs can be captured on Au electrode surface produced a significantly amplified signal by the reduction of hydrogen peroxide (H2O2) in the presence of redox mediator hydroquinone (HQ). The prepared electrochemical immunosensors showed excellent detection limit (1.2 pg mL-1), wide liner range (0.00975-5 ng mL-1) and good selectivity, demonstrating the promising potential application for the clinical diagnosis of HIV infection.

目錄
摘要 i
Abstract ii
圖目錄 v
表目錄 viii
第一章 緒論 1
1.1 前言 1
1.2 抗體抗原 2
1.2.1 抗體抗原簡介 2
1.2.2 抗體的基本結構 2
1.2.3 人類免疫缺陷病毒 3
1.3 生物感測器 6
1.3.1 生物感測器之介紹 6
1.3.2 生物感測器之基本構造與組成 7
1.3.3 生物感測器之分類 14
1.4 免疫感測器 18
1.4.1 光學式人類免疫缺陷病毒免疫感測器 18
1.4.2 質量感應式人類免疫缺陷病毒免疫感測器 20
1.4.3 電致化學發光式人類免疫缺陷病毒免疫感測器 21
1.4.4 電化學式人類免疫缺陷病毒免疫感測器 22
1.5 石墨烯量子 26
1.5.1 石墨烯量子點之簡介 26
1.5.2 石墨烯量子點之製備 28
1.5.2.1由上而下(top-down)： 29
1.6 電化學方法 36
1.6.1 循環伏安法(cyclic voltammetry, CV) 36
1.6.2 微分脈衝伏安法 (differential pulse voltammetry, DPV) 38
1.6.3 交流阻抗法(AC impedance) 39
第二章 實驗方法與步驟 41
2.1 實驗藥品 41
2.2 實驗儀器 42
2.3 實驗步驟 43
2.3.1 電極前處理 43
2.3.2 石墨烯量子點合成 44
2.3.3 石墨烯量子點與抗體之接合 45
2.3.4 人類免疫缺陷病毒免疫感測器之製備 45
第三章 結果與討論 47
3.1 石墨烯量子點之探討 48
3.1.1 石墨烯量子點之鑑定 48
3.1.2 石墨烯量子點與抗體接合之鑑定 49
3.2 利用電化學方法偵測人類免疫缺陷病毒之探討 52
3.2.1 人類免疫缺陷病毒免疫感測器之電化學特性 52
3.2.2 人類免疫缺陷病毒免疫感測器之時間探討 55
3.2.3 人類免疫缺陷病毒免疫感測器之pH值探討 60
3.2.4 人類免疫缺陷病毒免疫感測器之校正曲線探討 61
3.2.5 人類免疫缺陷病毒免疫感測器之選擇性探討 64
3.2.6 人類免疫缺陷病毒免疫感測器之穩定性探討 65
第四章 結論與未來展望 66
4.1 結論 66
4.2 未來展望 66
第五章 參考文獻 67


圖目錄
圖 1.2.1 抗體分子的基本結構 2
圖 1.2.2 抗體分子的方向性 3
圖 1.2.3 人類免疫缺陷病毒之結構示意圖 4
圖 1.2.4 人類免疫缺陷病毒免疫偵測器製備流程示意圖 5
圖 1.2.5 本國籍感染人類免疫缺乏病毒者趨勢 5
圖 1.3.1 生物感測器之基本構造 7
圖 1.3.2 光學式生物感測器 9
圖 1.3.3 壓電式生物感測器 10
圖 1.3.4 熱感式生物感測器 10
圖 1.3.5 電導式生物感測器 12
圖 1.3.6 電位式生物感測器 13
圖 1.3.7 電流式生物感測器 14
圖 1.3.8 酵素反應式生物感測器示意圖 15
圖 1.3.9 DNA生物感測器示意圖 16
圖 1.3.10 (a)競爭型及(b)非競爭型免疫分析示意圖 17
圖 1.3.11 免疫生物感測器示意圖 17
圖 1.4.1 (a)螢光式免疫生物感測器示意圖(b)螢光探針示意圖 18
圖 1.4.2 比色式免疫生物感測器 19
圖 1.4.3 質量感應式免疫生物感測器(a)實驗流程圖及(b)示意圖 20
圖 1.4.4 電致化學發光式免疫生物感測器 21
圖 1.4.5 免標記式免疫生物感測器之示意圖 22
圖 1.4.6 免標記式免疫生物感測器之實驗流程示意圖 23
圖 1.4.7 三明治式免疫感測器示意圖 24
圖 1.4.8 三明治式免疫感測器之DPV圖 24
圖 1.4.9 三明治式免疫感測器示意圖 25
圖 1.4.10 三明治式免疫感測器之DPV圖及檢量線 25
圖 1.5.1 石墨烯量子點製備示意圖 27
圖 1.5.2 石墨烯量子點表面官能基改質示意圖 27
圖 1.5.3由上而下(top-down)和由下而上(bottom-up)的示意圖 28
圖 1.5.4以水熱法製備石墨烯量子點之 (a) TEM (b)粒徑分佈(c)在365nm UV光下之螢光呈色 29
圖 1.5.5水熱法製備石墨烯量子點之示意圖 29
圖 1.5.6電化學法製備石墨烯量子點之(a) UV-vis光譜(b)PL光譜及石墨烯量子點在365 nm UV光下之螢光 (c) HRTEM及(d)石墨烯量子點之sp2結構。
30
圖 1.5.7電化學法製備石墨烯量子點示意圖 31
圖 1.5.8（a）利用油胺剝離石墨裂解製成石墨烯量子點示意圖 （b）不同油胺濃度導致不同粒徑石墨烯量子點之TEM圖（e）在365 nm紫外燈下，不同各種尺寸的GQDs的發光顏色 32
圖 1.5.9 溶液化學法製備石墨烯量子點之(a)流程圖(b)控制石墨烯量子點大小得到168、132 和170 個共軛碳原子。 33
圖 1.5.10(a)檸檬酸製備石墨烯量子點或氧化石墨烯示意圖(b)石墨烯量子點在365 nm UV光下之螢光。 34
圖 1.5.11 溶液化學法製備石墨烯量子點之(a)流程圖(b)控制石墨烯量子點大小得到168、132 和170 個共軛碳原子 35
圖 1.6.1典型之循環伏安法波形示意圖 37
圖 1.6.2典型之循環伏安圖 38
圖 1.6.3微分脈衝伏安法波形示意圖 38
圖 1.6.4輸入電流與電壓之應答關係圖 39
圖 1.6.5等效電路圖 40
圖 1.6.6 Nyquist plot之示意圖 40
圖 2.3.1使用循環伏安法進行電極前處理 43
圖 2.3.2 (a)石墨烯量子點溶液(b)石墨烯量子點在365 nm UV光下之藍色螢光 44
圖 2.3.3石墨烯量子點與人類免疫缺陷病毒抗體修飾辣根過氧化酶接合示意圖 45
圖 2.3.4人類免疫缺陷病毒免疫感測器製作步驟示意圖 46
圖 3.1.1石墨烯量子點之UV/Vis圖 48
圖 3.1.2石墨烯量子點之TEM圖 49
圖 3.1.3 HRP-HIV-Ab2-GQDs之UV/Vis圖 50
圖 3.1.4 HRP-HIV-Ab2-GQDs之TEM圖 50
圖 3.1.5 (a) GQDs 與(b) HRP-HIV-Ab2-GQDs之FTIR圖 51
圖 3.2.1不同修飾電極之循環伏安圖 (a)未修飾電極，(b)mpa修飾電極，(c)mpa/Ab1修飾電極，(d)mpa/Ab1/Ag修飾電極。(溶液為含有1mM HQ和1 mM H2O2的PBS溶液，掃描速率為50 mV/s )
53
圖 3.2.2不同修飾電極之循環伏安圖 (a) mpa/Ab1/Ag/HRP-HIV-Ab2-GQDs修飾電極(b) mpa/Ab1/Ag/HRP-HIV-Ab2修飾電極。(溶液為含有2 mM HQ和2 mM H2O2的PBS溶液，掃描速率為50 mV/s )
53
圖 3.2.3不同修飾電極之電阻抗圖 (a)未修飾電極，(b)mpa修飾電極，(c)mpa/Ab1修飾電極，(d)mpa/Ab1/Ag修飾電極，(e)mpa/Ab1/Ag/HRP-HIV-Ab2-GQDs修飾電極。(溶液為含有5 mM [Fe(CN)6]3-和5 mM [Fe(CN)6]4-和0.1 M KCl的0.1 M PBS溶液，振幅 10 mV，掃瞄範圍為100000赫茲至0.01赫茲。)
54
圖 3.2.4圓盤狀電極之等效電路圖 55
圖3.2.5未修飾電極浸泡mpa溶液(a)0 hr、(b)1 hr、(c)2 hr、(d)3 hr及(e)4 hr (f)5 hr的微分脈衝伏安圖 56
圖3.2.6電極浸泡mpa溶液的時間與電流差關係圖 56
圖 3.2.7 mpa修飾電極浸泡Ab1溶液(a)1 hr、(b)2 hr、(c)3 hr及(d)4 hr (e)5 hr的微分脈衝伏安圖
57
圖 3.2.8電極浸泡Ab1溶液的時間與電流差關係圖 57
圖 3.2.9 MPA/Ab1修飾電極浸泡Ag溶液(a)1 hr、(b)2 hr、(c)3 hr及(d)4 hr的微分脈衝伏安圖
58
圖 3.2.10電極浸泡Ag溶液的時間與電流差關係圖 58
圖 3.2.11 MPA/Ab1/Ag修飾電極浸泡HRP-HIV-Ab2-GQDs溶液(a)1 hr、(b)2 hr、(c)3 hr及(d)4 hr的微分脈衝伏安圖 59
圖 3.2.12電極浸泡HRP-HIV-Ab2-GQDs溶液的時間與電流差關係圖 59
圖 3.2.13 (a)pH 4.0、(b)pH 5.0、(c)pH 6.0、(d)pH 7.0及(e)pH 8.0的0.1 M PBS溶液含有2 mM HQ和2 mM H2O2下所測之微分脈衝伏安圖 60
圖 3.2.14溶液pH值與電流差關係圖 61
圖 3.2.15 不同濃度人類免疫缺陷病毒抗原之微分脈衝伏安圖 62
圖 3.2.16 利用微分脈衝伏安法偵測不同濃度人類免疫缺陷病毒抗原之校正曲線 62
圖 3.2.17 利用微分脈衝伏安法偵測不同抗原濃度與干擾物之電流差直方圖 64
圖 3.2.18 利用微分脈衝伏安法做穩定性測試之電流差與時間關係圖 65

 
表目錄
表 1.3.1生物辨識元件分類表 8
表 3.2.1各種人類免疫缺陷病毒免疫感測器之效能參數比較 .. 63

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